This is a competitive renewal application of NIDA grant DA000564-34. Since opioid receptors have been identified to be members of the rhodopsin subfamily of GPCRs, the general mechanism for GPCR desensitization has been applied to account for in vivo opiate tolerance. In this mechanism, opioid receptors are phosphorylated by GRKs in the presence of agonists and beta-arrestin is recruited to the receptor vicinity resulting in the blunting of the signals. Such mechanism is supported by studies with beta-arrestin knockout mice in which morphine tolerance was attenuated. However, a direct correlation between receptor phosphorylation and desensitization could not be demonstrated. Nevertheless, it remains our central hypothesis that covalent modification of opioid receptor and the signaling complex, or receptosome, during chronic agonist treatment is the key for the eventual manifestation of opiate tolerance and dependence. Therefore, in the proposed studies, we will continue our on-going studies to investigate the role of MOR phosphorylation on morphine tolerance response. We will determine the protein kinases involved in the phosphorylation of the consensus motif TXXXPS within the receptor. We will re-determine the agonist-dependent MOR phosphorylation sites by mass spectrometry analyses of purified receptor. We will validate our observations in clonal cell models with primary neuronal cultures. We will investigate the role of receptor phosphorylation in morphine tolerance by the use of GRK null mice together with inhibitors of protein kinases involved in MOR phosphorylation. The role of receptor phosphorylation in morphine tolerance will also be investigated by restoration of morphine acute and chronic responses in MOR null mice with adenovirus carrying the wild type and phosphorylation receptor mutants. In addition to receptor desensitization, our preliminary data also suggested that Src activation during chronic morphine treatment could be the basis for adenylyl cyclase (AC) superactivation. Since beta-arrestin has been implicated in Src activation, receptor phosphorylation could be the trigger for such event. Thus, we will examine the mechanism for and the role of beta-arrestin in Src activation. We will investigate the consequence of Src activation on AC superactivation, and will identify the cellular targets of Src with mass spectrometry analysis. The role of Src activation in the in vivo morphine dependence and withdrawal responses will be established by the use of Src inhibitor PP2 and inducible siRNA approach. All these studies will allow us to elucidate the molecular components involved in opiate tolerance and dependence.